3-iodotyrosine fluorescent proteins for improved expansion microscopy

Area of Science:

• Biotechnology
• Microscopy
• Molecular Biology

Background:

• Expansion microscopy (ExM) achieves nanoscale resolution but suffers from fluorescent protein (FP) signal degradation.
• Current ExM detection methods rely on immunostaining or other labeling techniques, limiting direct protein visualization.
• Signal loss during ExM hinders the full potential of protein-based imaging.

Purpose of the Study:

• To develop a novel fluorescent protein suitable for robust signal retention during expansion microscopy.
• To overcome the limitations of signal degradation in current fluorescent protein-based ExM approaches.
• To enable reliable protein visualization in ExM using genetically encoded tags.

Main Methods:

• Modification of superfolder green fluorescent protein (GFP) by substituting tyrosine with 3-iodotyrosine in its chromophore.
• Application of modified GFP in expansion microscopy experiments using zebrafish embryos.
• Assessment of signal retention and preservation of nanoscale resolution during the expansion process.

Main Results:

• 3-iodotyrosine-modified GFP demonstrated significantly improved signal retention during expansion microscopy.
• The modified FP showed enhanced stability, particularly during the formaldehyde-dependent anchoring step.
• This modification offers better tolerance to acidic conditions encountered during ExM processing.
• Successful application in zebrafish embryos, validating its utility in a biological system.

Conclusions:

• 3-iodotyrosine modification is a viable strategy to enhance FP stability for expansion microscopy.
• This innovation facilitates reliable, protein-based expansion microscopy, expanding its applicability.
• Opens new avenues for high-resolution imaging of proteins in biological samples.